BRD4 is a chromatin-binding protein with kinase and acetyl transferase activities that links chromatin structure and transcription. BRD4 acetylation of nucleosomal histones leads to chromatin decompaction and increased transcription. It has been implicated in autoimmune diseases and cancer. BRD4 is necessary for embryonic development: germline deletion is lethal. We examined BRD4's role in thymocyte differentiation. BRD4 deletion at the DN stage of thymocyte differentiation resulted in a 70% reduction in total thymocyte number due to impaired transition from DN to DP stages. The decrease in total thymocytes resulted from decreases in DP; the proportion of DN4 and ISP cells was increased. Importantly, BRD4-/- ISP thymocytes do not divide or become DP, althoughno TCRb rearrangement defect was observed. In contrast, the transition from DP to SP thymocytes occurred in the absence of BRD4. BRD4-/- CD4 and CD8 SPs o exited the thymus and colonized peripheral organs. Although CD4+ T cells were generated in its absence, BRD4 deletion ablated Treg differentiation. Thus, BRD4 plays critical roles at various stages of thymocyte differentiation. Consistent with its role as a chromatin remodeler, BRD4-/- thymocytes have significantly reduced levels of acetylated histones necessary for chromatin decompaction and transcription.